1 Running head: Steryl glycoside mutants Name: Seth DeBolt ...

Relative gene expression of proUGT80A2::GUS was observed in a patchy distribution in
cauline leaf epidermal cells, stomata, pollen, around the base of siliques and in the
stamen (Fig S4). Relative gene expression of proUGT80B1::GUS was primarily
observed in leaves, seedlings, around the apical tip of cotyledons and developing seeds
(Fig S5). Characterization of the GUS staining pattern in embryos revealed that
expression was strongest around the apical tip of the cotyledons and at the root apex.
Strong GUS expression was also apparent around the seed coat epidermal cell boundaries
and in the central columella, but not in the trough (Fig S5). Taken together the results
indicate that UGT80A2 and UGT80B1 mRNAs are found in distinct expression domains
within the plant. UGT80B1 was uniquely expressed in the seed coat and in the cotyledons
of the embryo, consistent with its mutant phenotype related to these tissues. The
promoter fusion expression results for both genes are largely consistent with expression
analysis performed using available microarray data for Arabidopsis (available online
through The Arabidopsis Information Resource; https://3.met.genevestigator.com/).
Mutations in UGT80A2 and USG80B1 genes result in reduced seed size, transparent
testa and salt uptake phenotypes
Visual inspection revealed a lightened seed coat hue in ugt80B1 and in the double
mutant as well as a dramatic reduction in seed size compared to wild-type (Fig 4a, Fig
S6). The light colored testa phenotype in the UGT80B1 mutant was consistent with being
a transparent testa mutant and this was confirmed by allelism tests to transparent testa
15 (data not shown) (Focks et al., 1999). The color of seed from ugt80A2 mutants did
not appear lightened to the same degree as ugt80B1, however ugt80A2 seeds displayed a
measurable reduction in size (Fig 4a, Fig S6).
A series of histochemical and microscopy analyses were performed to deduce
possible reasons for the increased permeability of the ugt80B1 mutant. When seeds were
incubated in a solution of tetrazolium salts, ugt80B1 and the double mutant seed were
found to be highly sensitive to salt uptake (Fig 4b). Wild-type and ugt80A2 seeds
absorbed small amount of salt at the hilum region, but ugt80B1 seeds were unable to limit
uptake and the entire embryo became stained with formazan dye (a tetrazolium reduction
product) (Fig 4b). The salt-uptake phenotype of ugt80B1 mutants was rescued by
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